Biological and Chemical Characterization of Musa paradisiaca Leachate.

There is a growing demand for molecules of natural origin for biocontrol and biostimulation, given the current trend away from synthetic chemical products. Leachates extracted from plantain stems were obtained after biodegradation of the plant material. To characterize the leachate, quantitative determinations of nitrogen, carbon, phosphorus, and cations (K+, Ca2+, Mg2+, Na+), Q2/4, Q2/6, and Q4/6 absorbance ratios, and metabolomic analysis were carried out. The potential role of plantain leachates as fungicide, elicitor of plant defense, and/or plant biostimulant was evaluated by agar well diffusion method, phenotypic, molecular, and imaging approaches. The plant extracts induced a slight inhibition of fungal growth of an aggressive strain of Colletotrichum gloeosporioides, which causes anthracnose. Organic compounds such as cinnamic, ellagic, quinic, and fulvic acids and indole alkaloid such as ellipticine, along with some minerals such as potassium, calcium, and phosphorus, may be responsible for the inhibition of fungal growth. In addition, jasmonic, benzoic, and salicylic acids, which are known to play a role in plant defense and as biostimulants in tomato, were detected in leachate extract. Indeed, foliar application of banana leachate induced overexpression of LOXD, PPOD, and Worky70-80 genes, which are involved in phenylpropanoid metabolism, jasmonic acid biosynthesis, and salicylic acid metabolism, respectively. Leachate also activated root growth in tomato seedlings. However, the main impact of the leachate was observed on mature plants, where it caused a reduction in leaf area and fresh weight, the remodeling of stem cell wall glycopolymers, and an increase in the expression of proline dehydrogenase.

LAM2: An Unusual Laminaran Structure for a Novel Plant Elicitor Candidate.

Laminarans are of interest because they have been shown to induce various immune responses in animals and plants. These ß-D-glucans differ from each other by their branching rate, which is possibly responsible for their biological activities. In the present study, we characterized a laminaran fraction extracted from Laminaria hyperborea and named LAM2 using sugar composition and structural analyses (NMR). Then, we evaluated its activity as a potential plant elicitor in vitro on tomato seedlings using gene expression analysis and cell wall immunofluorescence labeling. Our study showed that LAM2 isolated from L. hyperborea is a succinylated laminaran which significantly enhanced the plant defense of tomato seedlings and induced cell wall modifications, suggesting a higher elicitor activity than the laminaran standard extracted from Laminaria digitata.

Living yeast-based biostimulants: different genes for the same results?

Nowadays, many products are available in the plant biostimulants market. Among them, living yeast-based biostimulants are also commercialized. Given the living aspect of these last products, the reproducibility of their effects should be investigated to ensure end-users' confidence. Therefore, this study aimed to compare the effects of a living yeast-based biostimulant between two different soybean cultures. These two cultures named C1 and C2 were conducted on the same variety and soil but in different locations and dates until the VC developmental stage (unifoliate leaves unrolled), with Bradyrhizobium japonicum (control and Bs condition) and with and without biostimulant coating seed treatment. The foliar transcriptomic analysis done first showed a high gene expression difference between the two cultures. Despite this first result, a secondary analysis seemed to show that this biostimulant led to a similar pathway enhancement in plants and with common genes even if the expressed genes were different between the two cultures. The pathways which seem to be reproducibly impacted by this living yeast-based biostimulant are abiotic stress tolerance and cell wall/carbohydrate synthesis. Impacting these pathways may protect the plant from abiotic stresses and maintain a higher level of sugars in plant.

Black Poplar (Populus nigra L.) Root Extracellular Trap, Structural and Molecular Remodeling in Response to Osmotic Stress.

The root extracellular trap (RET) consists of root-associated, cap-derived cells (root AC-DCs) and their mucilaginous secretions, and forms a structure around the root tip that protects against biotic and abiotic stresses. However, there is little information concerning the changes undergone by the RET during droughts, especially for tree species. Morphological and immunocytochemical approaches were used to study the RET of black poplar (Populus nigra L.) seedlings grown in vitro under optimal conditions (on agar-gelled medium) or when polyethylene glycol-mediated (PEG6000-infused agar-gelled medium) was used to mimic drought conditions through osmotic stress. Under optimal conditions, the root cap released three populations of individual AC-DC morphotypes, with a very low proportion of spherical morphotypes, and equivalent proportions of intermediate and elongated morphotypes. Immunolabeling experiments using anti-glycan antibodies specific to cell wall polysaccharide and arabinogalactan protein (AGP) epitopes revealed the presence of homogalacturonan (HG), galactan chains of rhamnogalacturonan-I (RG-I), and AGPs in root AC-DC cell walls. The data also showed the presence of xylogalacturonan (XGA), xylan, AGPs, and low levels of arabinans in the mucilage. The findings also showed that under osmotic stress conditions, both the number of AC-DCs (spherical and intermediate morphotypes) and the total quantity of mucilage per root tip increased, whereas the mucilage was devoid of the epitopes associated with the polysaccharides RG-I, XGA, xylan, and AGPs. Osmotic stress also led to reduced root growth and increased root expression of the P5CS2 gene, which is involved in proline biosynthesis and cellular osmolarity maintenance (or preservation) in aerial parts. Together, our findings show that the RET is a dynamic structure that undergoes pronounced structural and molecular remodeling, which might contribute to the survival of the root tip under osmotic conditions.

Elicitation of Roots and AC-DC with PEP-13 Peptide Shows Differential Defense Responses in Multi-Omics.

The root extracellular trap (RET) has emerged as a specialized compartment consisting of root AC-DC and mucilage. However, the RET's contribution to plant defense is still poorly understood. While the roles of polysaccharides and glycoproteins secreted by root AC-DC have started to be elucidated, how the low-molecular-weight exudates of the RET contribute to root defense is poorly known. In order to better understand the RET and its defense response, the transcriptomes, proteomes and metabolomes of roots, root AC-DC and mucilage of soybean (Glycine max (L.) Merr, var. Castetis) upon elicitation with the peptide PEP-13 were investigated. This peptide is derived from the pathogenic oomycete Phytophthora sojae. In this study, the root and the RET responses to elicitation were dissected and sequenced using transcriptional, proteomic and metabolomic approaches. The major finding is increased synthesis and secretion of specialized metabolites upon induced defense activation following PEP-13 peptide elicitation. This study provides novel findings related to the pivotal role of the root extracellular trap in root defense.

Dynamic imaging of cell wall polysaccharides by metabolic click-mediated labeling of pectins in living elongating cells.

Protein tracking in living plant cells has become routine with the emergence of reporter genes encoding fluorescent tags. Unfortunately, this imaging strategy is not applicable to glycans because they are not directly encoded by the genome. Indeed, complex glycans result from sequential additions and/or removals of monosaccharides by the glycosyltransferases and glycosidases of the cell's biosynthetic machinery. Currently, the imaging of cell wall polymers mainly relies on the use of antibodies or dyes that exhibit variable specificities. However, as immunolocalization typically requires sample fixation, it does not provide access to the dynamics of living cells. The development of click chemistry in plant cell wall biology offers an alternative for live-cell labeling. It consists of the incorporation of a carbohydrate containing a bio-orthogonal chemical reporter into the target polysaccharide using the endogenous biosynthetic machinery of the cell. Once synthesized and deposited in the cell wall, the polysaccharide containing the analog monosaccharide is covalently coupled to an exogenous fluorescent probe. Here, we developed a metabolic click labeling approach which allows the imaging of cell wall polysaccharides in living and elongating cells without affecting cell viability. The protocol was established using the pollen tube, a useful model to follow cell wall dynamics due to its fast and tip-polarized growth, but was also successfully tested on Arabidopsis root cells and root hairs. This method offers the possibility of imaging metabolically incorporated sugars of viable and elongating cells, allowing the study of the long-term dynamics of labeled extracellular polysaccharides.

Faba bean root exudates alter pea root colonization by the oomycete Aphanomyces euteiches at early stages of infection.

Aphanomyces euteiches is an oomycete pathogen that causes the pea root rot. We investigated the potential role of early belowground defense in pea (susceptible plant) and faba bean (tolerant plant) at three days after inoculation. Pea and faba bean were inoculated with A. euteiches zoospores. Root colonization was examined. Root exudates from pea and faba bean were harvested and their impact on A. euteiches development were assessed by using in vitro assays. A. euteiches root colonization and the influence of the oomycete inoculation on specialized metabolites patterns and arabinogalactan protein (AGP) concentration of root exudates were also determined. In faba bean root, A. euteiches colonization was very low as compared with that of pea. Whereas infected pea root exudates have a positive chemotaxis index (CI) on zoospores, faba bean exudate CI was negative suggesting a repellent effect. While furanoacetylenic compounds were only detected in faba bean exudates, AGP concentration was specifically increased in pea.This work showed that early in the course of infection, host susceptibility to A. euteiches is involved via a plant-species specific root exudation opening new perspectives in pea root rot disease management.

Chromosomal scale assembly of parasitic wasp genome reveals symbiotic virus colonization.

Endogenous viruses form an important proportion of eukaryote genomes and a source of novel functions. How large DNA viruses integrated into a genome evolve when they confer a benefit to their host, however, remains unknown. Bracoviruses are essential for the parasitism success of parasitoid wasps, into whose genomes they integrated ~103 million years ago. Here we show, from the assembly of a parasitoid wasp genome at a chromosomal scale, that bracovirus genes colonized all ten chromosomes of Cotesia congregata. Most form clusters of genes involved in particle production or parasitism success. Genomic comparison with another wasp, Microplitis demolitor, revealed that these clusters were already established ~53 mya and thus belong to remarkably stable genomic structures, the architectures of which are evolutionary constrained. Transcriptomic analyses highlight temporal synchronization of viral gene expression without resulting in immune gene induction, suggesting that no conflicts remain between ancient symbiotic partners when benefits to them converge.

New Insights into Plant Extracellular DNA. A Study in Soybean Root Extracellular Trap.

exDNA is found in various organisms, including plants. However, plant exDNA has thus far received little attention related to its origin and role in the RET (root extracellular trap). In this study, we performed the first high-throughput genomic sequencing of plant exDNA from a Fabaceae with worldwide interest: soybean (Glycine max (L.) Merr.). The origin of this exDNA was first investigated in control condition, and the results show high-coverage on organelles (mitochondria/plastid) DNA relative to nuclear DNA, as well as a mix of coding and non-coding sequences. In the second part of this study, we investigated if exDNA release was modified during an elicitation with PEP-13 (a peptide elicitor from oomycete genus Phytophthora). Our results show that treatment of roots with PEP-13 does not affect the composition of exDNA.

Heterodimeric Insecticidal Peptide Provides New Insights into the Molecular and Functional Diversity of Ant Venoms.

Ants use venom for predation, defense, and communication; however, the molecular diversity, function, and potential applications of ant venom remains understudied compared to other venomous lineages such as arachnids, snakes and cone snails. In this work, we used a multidisciplinary approach that encompassed field work, proteomics, sequencing, chemical synthesis, structural analysis, molecular modeling, stability studies, and in vitro and in vivo bioassays to investigate the molecular diversity of the venom of the Amazonian Pseudomyrmex penetrator ants. We isolated a potent insecticidal heterodimeric peptide Δ-pseudomyrmecitoxin-Pp1a (Δ-PSDTX-Pp1a) composed of a 27-residue long A-chain and a 33-residue long B-chain cross-linked by two disulfide bonds in an antiparallel orientation. We chemically synthesized Δ-PSDTX-Pp1a, its corresponding parallel AA and BB homodimers, and its monomeric chains and demonstrated that Δ-PSDTX-Pp1a had the most potent insecticidal effects in blowfly assays (LD50 = 3 nmol/g). Molecular modeling and circular dichroism studies revealed strong α-helical features, indicating its cytotoxic effects could derive from cell membrane pore formation or disruption. The native heterodimer was substantially more stable against proteolytic degradation (t 1/2 = 13 h) than its homodimers or monomers (t 1/2 < 20 min), indicating an evolutionary advantage of the more complex structure. The proteomic analysis of Pseudomyrmex penetrator venom and in-depth characterization of Δ-PSDTX-Pp1a provide novel insights in the structural complexity of ant venom and further exemplifies how nature exploits disulfide-bond formation and dimerization to gain an evolutionary advantage via improved stability, a concept that is highly relevant for the design and development of peptide therapeutics, molecular probes, and bioinsecticides.

Root Border Cells and Mucilage Secretions of Soybean, Glycine Max (Merr) L.: Characterization and Role in Interactions with the Oomycete Phytophthora Parasitica.

Root border cells (BCs) and their associated secretions form a protective structure termed the root extracellular trap (RET) that plays a major role in root interactions with soil borne microorganisms. In this study, we investigated the release and morphology of BCs of Glycine max using light and cryo-scanning electron microscopy (SEM). We also examined the occurrence of cell-wall glycomolecules in BCs and secreted mucilage using immunofluorescence microscopy in conjunction with anti-glycan antibodies. Our data show that root tips released three populations of BCs defined as spherical, intermediate and elongated cells. The mechanism of shedding seemed to be cell morphotype-specific. The data also show that mucilage contained pectin, cellulose, extracellular DNA, histones and two hemicellulosic polysaccharides, xyloglucan and heteromannan. The latter has never been reported previously in any plant root secretions. Both hemicellulosic polysaccharides formed a dense fibrillary network embedding BCs and holding them together within the mucilage. Finally, we investigated the effect of the RET on the interactions of root with the pathogenic oomycete Phytophthora parasitica early during infection. Our findings reveal that the RET prevented zoospores from colonizing root tips by blocking their entry into root tissues and inducing their lysis.

A Novel In Vitro Tool to Study Cyst Nematode Chemotaxis.

This study presents a novel three-dimensional (3D) tool "3D in vitro choice" for chemotaxis assays with cyst nematodes. The original 3D in vitro choice was customized through digital printing. Freshly hatched second stage juveniles (J2s) of the cyst nematode Globodera pallida were used as the nematode model to illustrate chemo-orientation behavior in the 3D system. The efficiency and reliability of the 3D in vitro choice were validated with 2% Phytagel as navigation medium, in three biological assays and using tomato root exudates or potato root border cells and their associated mucilage as a positive attractant as compared with water. For each biological assay, J2s were hatched from the same population of a single generation glasshouse-cultured cysts. This novel easy to use and low-cost 3d-device could be a useful replacement to Petri dishes assays in nematode behavioral studies due to the ease of deposition of nematodes and test substances, coupled with its distinctive zones that allow for precision in choice making by the nematodes.

Effects of DEHP on the ecdysteroid pathway, sexual behavior and offspring of the moth Spodoptera littoralis.

Bis(2-ethylhexyl) phthalate (DEHP) is a widely produced plasticizer that is considered to act as an endocrine-disrupting chemical in vertebrates and invertebrates. Indeed, many studies have shown that DEHP alters hormonal levels, reproduction and behavior in vertebrates. Few studies have focused on the effects of DEHP on insects, although DEHP is found almost everywhere in their natural habitats, particularly in soils and plants. Here, we investigated the effects of DEHP on the sexual behavior and physiology of a pest insect, the noctuid moth Spodoptera littoralis. In this nocturnal species, olfaction is crucial for sexual behavior, and ecdysteroids at the antennal level have been shown to modulate sex pheromone detection by males. In the present study, larvae were fed food containing different DEHP concentrations, and DEHP concentrations were then measured in the adults (males and females). Hemolymphatic ecdysteroid concentrations, the antennal expression of genes involved in the ecdysteroid pathway (nuclear receptors EcR, USP, E75, and E78 and calmodulin) and sexual behavior were then investigated in adult males. The success and latency of mating as well as the hatching success were also studied in pairs consisting of one DEHP male and one uncontaminated female or one DEHP female and one uncontaminated male. We also studied the offspring produced from pairs involving contaminated females to test the transgenerational effect of DEHP. Our results showed the general downregulation of nuclear receptors and calmodulin gene expression associated with the higher concentrations of DEHP, suggesting peripheral olfactory disruption. We found some effects on male behavior but without an alteration of the mating rate. Effects on offspring mortality and developmental rates in the N + 1 generation were also found at the higher doses of DEHP. Taken together, the results of the study show for the first time that larval exposure to DEHP can induce delayed endocrine-disruptive effects in the adults of a terrestrial insect as well as effects on the next generation. To date, our study is also the first description of an impact of endocrine disrupter on olfaction in insects.

Towards the optimization of botanical insecticides research: Aedes aegypti larvicidal natural products in French Guiana.

Natural products have proven to be an immeasurable source of bioactive compounds. The exceptional biodiversity encountered in Amazonia, alongside a rich entomofauna and frequent interactions with various herbivores is the crucible of a promising chemodiversity. This prompted us to search for novel botanical insecticides in French Guiana. As this French overseas department faces severe issues linked to insects, notably the strong incidence of vector-borne infectious diseases, we decided to focus our research on products able to control the mosquito Aedes aegypti. We tested 452 extracts obtained from 85 species originating from 36 botanical families and collected in contrasted environments against an Ae. aegypti laboratory strain susceptible to all insecticides, and a natural population resistant to both pyrethroid and organophosphate insecticides collected in Cayenne for the most active of them. Eight species (Maytenus oblongata Reissek, Celastraceae; Costus erythrothyrsus Loes., Costaceae; Humiria balsamifera Aubl., Humiriaceae; Sextonia rubra (Mez) van der Werff, Lauraceae; Piper hispidum Sw., Piperaceae; Laetia procera (Poepp.) Eichl., Salicaceae; Matayba arborescens (Aubl.) Radlk., Sapindaceae; and Cupania scrobitulata Rich., Sapindaceae) led to extracts exhibiting more than 50% larval mortality after 48 h of exposition at 100 µg/mL against the natural population and were considered active. Selectivity and phytochemistry of these extracts were therefore investigated and discussed, and some active compounds highlighted. Multivariate analysis highlighted that solvents, plant tissues, plant family and location had a significant effect on mortality while light, available resources and vegetation type did not. Through this case study we highlighted that plant defensive chemistry mechanisms are crucial while searching for novel insecticidal products.

In vitro characterization of root extracellular trap and exudates of three Sahelian woody plant species.

MAIN CONCLUSION: Arabinogalactan protein content in both root extracellular trap and root exudates varies in three Sahelian woody plant species that are differentially tolerant to drought. At the root tip, mature root cap cells, mainly border cells (BCs)/border-like cells (BLCs) and their associated mucilage, form a web-like structure known as the "Root Extracellular Trap" (RET). Although the RET along with the entire suite of root exudates are known to influence rhizosphere function, their features in woody species is poorly documented. Here, RET and root exudates were analyzed from three Sahelian woody species with contrasted sensitivity to drought stress (Balanites aegyptiaca, Acacia raddiana and Tamarindus indica) and that have been selected for reforestation along the African Great Green Wall in northern Senegal. Optical and transmission electron microscopy show that Balanites aegyptiaca, the most drought-tolerant species, produces only BC, whereas Acacia raddiana and Tamarindus indica release both BCs and BLCs. Biochemical analyses reveal that RET and root exudates of Balanites aegyptiaca and Acacia raddiana contain significantly more abundant arabinogalactan proteins (AGPs) compared to Tamarindus indica, the most drought-sensitive species. Root exudates of the three woody species also differentially impact the plant soil beneficial bacteria Azospirillum brasilense growth. These results highlight the importance of root secretions for woody species survival under dry conditions.

Effects of bisphenol A on post-embryonic development of the cotton pest Spodoptera littoralis.

Endocrine-disrupting chemicals encompass a variety of chemicals that may interfere with the endocrine system and produce negative effects on organisms. Among them, bisphenol A is considered a major pollutant in numerous countries. The harmful effects of BPA on environmental and human health are intensely studied. However, the effects of BPA on terrestrial insects are still poorly investigated, despite that several plants can accumulate BPA in their tissues, leading to potential contamination of herbivorous insects. Here, we used the leafworm Spodoptera littoralis, a polyphagous species, to study BPA effects on post-embryonic development. We studied the effects of BPA ingestion at environmental doses (e.g., 0.01, 0.1, and 1 µg/g of BPA) and high doses (e.g., 25 µg/g) on larval weight and stage duration, pupal length and sex ratio. BPA effects were investigated in more detail during the last larval instar, a crucial period for preparing pupation and metamorphosis, which are under endocrine control. We monitored the haemolymph concentration of ecdysteroids, hormones controlling moult and metamorphosis, as well as the expression levels of several nuclear receptors involved in the ecdysteroid signalling pathway. Our integrative study showed that, upon exposure doses, BPA can induce various effects on the viability, developmental time, growth and sex ratio. These effects were correlated with a delay of the ecdysteroid peak during the last larval instar and a modification of expression of EcR, USP, E75AB, E75D and Br-c. We provide new evidence about the events that occur after BPA exposure in insect contaminated by food ingestion.

Root extracellular traps versus neutrophil extracellular traps in host defence, a case of functional convergence?

The root cap releases cells that produce massive amounts of mucilage containing polysaccharides, proteoglycans, extracellular DNA (exDNA) and a variety of antimicrobial compounds. The released cells - known as border cells or border-like cells - and mucilage secretions form networks that are defined as root extracellular traps (RETs). RETs are important players in root immunity. In animals, phagocytes are some of the most abundant white blood cells in circulation and are very important for immunity. These cells combat pathogens through multiple defence mechanisms, including the release of exDNA-containing extracellular traps (ETs). Traps of neutrophil origin are abbreviated herein as NETs. Similar to phagocytes, plant root cap-originating cells actively contribute to frontline defence against pathogens. RETs and NETs are thus components of the plant and animal immune systems, respectively, that exhibit similar compositional and functional properties. Herein, we describe and discuss the formation, molecular composition and functional similarities of these similar but different extracellular traps.

Xyloglucan and cellulose form molecular cross-bridges connecting root border cells in pea (Pisum sativum).

Pea (Pisum sativum) root cap releases a large number of living border cells that secrete abundant mucilage into the extracellular medium. Mucilage contains a complex mixture of polysaccharides, proteins and secondary metabolites important for its structure and function in defense. Unlike xyloglucan and cellulose, pectin and arabinogalactan proteins have been investigated in pea root and shown to be major components of border cell walls and mucilage. In this study, we investigated the occurrence of xyloglucan and cellulose in pea border cells and mucilage using cytochemical staining, immunocytochemistry and laser scanning confocal microscopy. Our data show that i) unlike cellulose, xyloglucan is highly present in the released mucilage as a dense fibrillary network enclosing border cells and ii) that xyloglucan and cellulose form molecular cross-bridges that tether cells and maintain them attached together. These findings suggest that secreted xyloglucan is essential for mucilage strengthening and border cell attachment and functioning.

Effects of DEHP on post-embryonic development, nuclear receptor expression, metabolite and ecdysteroid concentrations of the moth Spodoptera littoralis.

Di (2-ethylhexyl) phthalate (DEHP) is recognized in vertebrates as an Endocrine Disrupting Chemical (EDC). DEHP can alter steroid hormones production, development, reproduction and behavior in vertebrates. Only few studies investigated DEHP effects on insects. However, some recent studies on aquatic insects showed that DEHP could also act as an EDC by interfering with the signaling pathways of ecdysteroids, the main hormones involved in the control of insect post-embryonic development and physiology. The aim of the study was to investigate (1) the fate of DEHP within a terrestrial insect species by exposing larvae to food containing a wide range of DEHP concentrations and (2) the effects of this chemical on their post-embryonic development and metamorphosis, by using a multi-level approach. DEHP was shown to be present both in larvae and resulting stages, with higher concentrations in chrysalises and adults than in larvae. DEHP concentrations also decreased at the end of the last larval instar, suggesting the metabolic transformation or excretion of this chemical during this time. Only the two highest DEHP doses induced higher insect mortality, whereas low and intermediate concentrations increased larval food consumption without affecting body weight. Metabolic profiles showed that in control insects, the last three days before metamorphosis correspond to a metabolic transition, but with time-dependent changes in treated insects. Interestingly, DEHP treatments also alter both hemolymphatic ecdysteroid titers and expression levels of ecdysteroid response genes. These results confirm that DEHP can alter insect post-embryonic development and metamorphosis, by interfering with ecdysteroid pathways.